Two birds with one stone: human SIRPα nanobodies for functional modulation and in vivo imaging of myeloid cells

Signal-regulatory protein α (SIRPα) expressed by myeloid cells is of particular interest for therapeutic strategies targeting the interaction between SIRPα and the “don’t eat me” ligand CD47 and as a marker to monitor macrophage infiltration into tumor lesions. To address both approaches, we developed a set of novel human SIRPα (hSIRPα)–specific nanobodies (Nbs). We identified high-affinity Nbs targeting the hSIRPα/hCD47 interface, thereby enhancing antibody-dependent cellular phagocytosis. For non-invasive in vivo imaging, we chose S36 Nb as a non-modulating binder. By quantitative positron emission tomography in novel hSIRPα/hCD47 knock-in mice, we demonstrated the applicability of 64Cu-hSIRPα-S36 Nb to visualize tumor infiltration of myeloid cells. We envision that the hSIRPα-Nbs presented in this study have potential as versatile theranostic probes, including novel myeloid-specific checkpoint inhibitors for combinatorial treatment approaches and for in vivo stratification and monitoring of individual responses during cancer immunotherapies.


Introduction
During tumor development, there is a continuous exchange between malignant cells, neighboring parenchymal cells, stromal cells, and immune cells.Together with the extracellular matrix and soluble mediators, these cells constitute the tumor microenvironment (TME).The composition of the immune infiltrate within the TME largely determines cancer progression and sensitivity to immunotherapies (1).Myeloid cells are known to regulate T-cell responses, thereby bridging innate and adaptive immunity (2)(3)(4).Tumor cells further utilize myeloid cells to create a pro-tumorigenic milieu by exploiting their ability to produce immune-regulating mediators (e.g., interleukin-6 and tumor necrosis factor), growth factors influencing tumor proliferation and vascularization (e.g., transforming growth factor-b and vascular endothelial growth factor), as well as matrix-degrading enzymes (e.g., matrix metalloproteinases) (5).Within the myeloid cell population, tumorassociated macrophages (TAMs) are highly abundant, and widely varying densities of up to 50% of the tumor mass are observed (6).At the same time, depending on their polarization state, TAMs exhibit partially opposing effects either as key drivers for tumor progression or by exerting potent antitumor activity (7,8).Consequently, monitoring tumor infiltration of TAMs is of great importance for patient stratification and companion diagnostic (9)(10)(11), and targeted recruitment or activation of anti-tumor TAMs opens new strategies to achieve persisting anti-tumor immune responses (12).
In this context, the myeloid-specific immune checkpoint signalregulatory protein a (SIRPa), expressed by monocytes, macrophages, dendritic cells, and neutrophils (13,14), represents an interesting theranostic target.Interaction with its ligand CD47, a "marker of self" virtually expressed by all cells of the body, mediates a "don't eat me" signal that inhibits phagocytosis, and prevents subsequent autoimmune responses.Exploiting this physiological checkpoint, tumor cells often upregulate CD47 and thereby escape recognition and removal by macrophages (15,16).Similarly, enhanced expression of SIRPa by intratumoral monocytes/ macrophages has recently been shown to be associated with poorer survival in follicular lymphoma, colorectal cancer, intrahepatic cholangiocarcinoma, and esophageal cancer (17)(18)(19).
To address the potential as a biomarker and immune modulating target, we generated human SIRPa (hSIRPa)-specific nanobodies (Nbs) for diagnostic and potential therapeutic applications.Nbs are single-domain antibodies derived from heavy-chain antibodies of camelids (20,21) and have emerged as versatile biologicals for therapeutic as well as diagnostic purposes (22)(23)(24).Compared with conventional antibodies, Nbs exhibit superior characteristics concerning chemical stability, solubility, and tissue penetration due to their small size and compact folding (20).Following a binary screening strategy, in-depth biochemical characterization, epitope mapping, and functional studies, we identified two hSIRPa-Nb subsets that either block the hSIRPa/ hCD47 interface or serve as inert probes for molecular imaging.

Selection of high-affinity anti-human SIRPa nanobodies
To generate Nbs against hSIRPa that can be used either as probes for diagnostic imaging or to modulate interaction with human CD47, we immunized an alpaca (Vicugna pacos) with the recombinant extracellular portion of hSIRPa and established an Nb phagemid library (2 × 10 7 clones).This Nb library was subjected to phage display-based selection campaigns targeting either the entire extracellular portion or exclusively domain 1 (D1) of hSIRPa (hSIRPaD1) to guide the selection of Nbs that specifically block the hSIRPa/hCD47 interaction.Sequencing of individual clones resulted in 14 unique hSIRPa Nbs with high diversity in the complementaritydetermining region 3 (CDR3) (Figure 1A; Supplementary Table 1).Nbs S7 to S36 were derived from the full-length hSIRPa screening, whereas Nbs S41 to S45 were identified as hSIRPaD1 binders.Individual Nbs were produced in Escherichia coli (E.coli) and isolated with high purity (Figure 1B).Folding stability of all Nbs was analyzed by differential scanning fluorimetry.For 12 out of the 14 Nb candidates, melting temperatures ranging from ~55°C to ~75°C without aggregation (Figures 1C, D; Supplementary Figure 1A) were determined, whereas affinity measurements against recombinant hSIRPa by biolayer interferometry (BLI) revealed K D values between ~0.12 nM and ~27 nM for 11 out of the 12 Nbs (Figures 1C, D; Supplementary Figure 1B).In addition, live-cell immunofluorescence staining of U2OS -Human Bone Osteosarcoma Epithelial Cells stably expressing full-length hSIRPa showed that all selected Nbs recognize hSIRPa localized at the plasma membrane (Figure 1E; Supplementary Figure 2A).

Domain mapping of hSIRPa Nbs
Whereas Nbs targeting hSIRPaD1 have a higher chance to block interaction with CD47, Nbs targeting domain D2 or D3 (hSIRPaD2 and hSIRPaD3) might be functionally inert, which is preferable for diagnostic approaches.Thus, we assessed domain specificity using U2OS cells expressing the individual domains of hSIRPa by immunofluorescence staining (Figure 2A, Supplementary Figure 2B).Eight Nbs (S12, S14, S17, S41, S42, S43, S44, and S45) stained hSIRPaD1, whereas Nbs S14 and S17 additionally stained hSIRPaD2.Five Nbs (S8, S21, S29, S33, and S36) revealed specific binding to hSIRPaD2, whereas only Nb S7 stained cells expressing hSIRPaD3.On the basis of their respective production yield, stability, affinity, domain specificity, and developability, we selected Nbs S7, S8, S12, S33, S36, S41, S44, and S45 for further characterization.To determine the diversity of epitopes recognized by this subset in more detail, we performed an epitope binning analysis using BLI (Figure 2B; Supplementary Figures 3A, B).On the basis of the results, we grouped the Nbs according to shared or overlapping epitopes and found two groups each for hSIRPaD1-targeting (Nbs S12 and S41 and Nbs S44 Biochemical characterization of hSIRPa Nbs.(A) Amino acid (aa) sequences of the complementarity-determining region (CDR) 3 from 14 unique hSIRPa Nbs (complete sequences shown in Supplementary Table 1) identified by a bidirectional screening strategy.Nbs S7 to S36 were selected against full-length hSIRPa and Nbs S41 to 45 against domain 1 of hSIRPa (hSIRPaD1).and S45) and hSIRPaD2-targeting (Nb S8 and Nbs S33 and S36) Nbs (Supplementary Figures 3A, B).

Binding of hSIRPa Nbs to primary human monocyte/macrophage cells
To evaluate whether our hSIRPa Nbs recognize endogenously expressed hSIRPa, we performed flow cytometry analysis of peripheral blood mononuclear cells (PBMCs) from three different donors (K1-K3).In addition to the monocyte/macrophage marker CD14, we also included the T-cell marker CD3 to evaluate potential recognition of T cells by hSIRPg-cross-reactive Nbs (Figure 3B).All hSIRPa Nbs, except S7, stained comparably on CD14 + PBMCs from all tested donors, whereas none of the Nbs stained CD3 + T cells (Figures 3B, C).
Considering our binary strategy to select hSIRPa Nbs (i) that are eligible to inhibit the hSIRPa/hCD47 interaction and (ii) as probes for positron emission tomography (PET)-based in vivo imaging of myeloid cells, we divided the identified Nbs into two subgroups.In the following, hSIRPaD1-targeting Nbs S12, S41, S44, and S45 were further investigated with respect to their inhibitory properties, and hSIRPaD2-targeting Nbs S8, S33, and S36 for their applicability as in vivo imaging probes.

hSIRPaD1 Nbs functionally block the interaction with hCD47
To evaluate potential inhibition of the interaction between hSIRPa and hCD47 (Figure 4A), we first performed a competitive BLI-based binding assay.As control, we used the anti-hSIRPa-blocking antibody KWAR23 (26).After incubation with Nb S44 or S45, binding of hSIRPa to CD47 was inhibited to a similar extent as upon addition of KWAR23; whereas only partial blocking was observed for S41, S12 showed no effect (Figure 4B).For functional analysis, we next tested the ability of hSIRPaD1-targeting Nbs to potentiate macrophage-mediated antibody-dependent cellular phagocytosis (ADCP) (Figure 4C).To this end, human monocyte-derived macrophages (MDMs) isolated from three different donors (K1-K3) were incubated with Epidermal Growth Factor Receptor (EGFR + ) expressing human colorectal adenocarcinoma DLD-1 cells preloaded with carboxyfluorescein diacetate succinimidyl ester (CFSE) alone or in the presence of the opsonizing EGFR-specific antibody cetuximab and hSIRPaD1targeting Nbs or the KWAR23 antibody as positive control.The degree of ADCP was determined on the basis of the detection of CD206 + CFSE + cells by flow cytometry analysis (Figure 4D).For all tested donors, macrophages exhibited minimal phagocytosis of DLD-1 cells without treatment, whereas phagocytic activity was significantly increased upon addition of cetuximab.In the presence of the hSIRPablocking antibody KWAR23, phagocytosis was further induced, which is in line with previous findings (26).Similarly, the hSIRPa-blocking Nbs S44 and S45 augmented ADCP in all three tested donors, whereas Nb S12 and S41 only revealed limited effect on macrophage-mediated phagocytosis (Figure 4E).From these results, we concluded that Nbs S44 and S45 represent promising candidates for further development as novel hSIRPa/CD47-inhibitory biologicals for potential therapeutic applications.

Inert hSIRPa-S36 Nb as lead candidate for non-invasive in vivo imaging
For the application as non-invasive PET tracer, immunologically inert hSIRPa Nbs are preferred.Thus, we selected Nbs S8, S33, and S36, which bind to hSIRPaD2, and performed a detailed analysis of the recognized epitopes by hydrogen-deuterium exchange mass spectrometry (HDX-MS).All selected Nbs recognized threedimensional epitopes within hSIRPaD2, which are spatially distant from the hSIRPa/hCD47 interface (Supplementary Table 2; Supplementary Figures 4A, B).In particular, S36 Nb showed the strongest deuteration protection (<−15%) for amino acid (aa) D163 to L187 and aa H202 to G207 of hSIRPa, whereas an additional slightly lower protection was observed for the region ranging from aa C140 to K153 (Supplementary Figures 4A, B).Considering its detailed epitope mapping, strong binding affinity, and good production yield, we selected S36 Nb as the lead candidate for imaging.
For radiolabeling, we conceived a novel protein engineering approach that enables site-specific chemical conjugation.We first adapted the sequence of the original S36 Nb by replacing all four lysine residues with arginine (hSIRPa-S36 K>R Nb) (Supplementary Figure 5A) and conjugated the chelator via isothiocyanate (p-NCSbenzyl-NODA-GA) to the remaining primary NH 2 -group at the Nterminus (Supplementary Figure 5A).The hSIRPa-S36 K>R Nb was producible with comparable yield and purity to the original version in E.coli (Supplementary Figure 5B) and efficient site-specific chelator conjugation (~96%) was confirmed by mass spectrometry.Most importantly, the hSIRPa-S36 K>R Nb showed comparable affinities and characteristics to the original S36 Nb (Supplementary Figures 5C-E).Finally, we examined the hSIRPa-S36 K>R Nb in the macrophage-dependent phagocytosis assay.Here, we observed a minor induction of macrophage-dependent phagocytosis that is comparable to the effect of the non-blocking Nb S12 (Supplementary Figure 5F; Figure 4E).From these results, we concluded that hSIRPa-S36 K>R Nb, represents a lead candidate suitable for non-invasive in vivo PET imaging of SIRPa expression.
To visualize the distribution of hSIRPa-positive cells in a tumorrelevant system, we employed a novel immunocompetent hSIRPa/ hCD47 KI mouse model (hSIRPa/hCD47 mice), expressing the extracellular domain of hSIRPa, and C57BL/6 wild-type (wt) mice as controls.In both models, tumors were generated by subcutaneous (s.c.) injection of hCD47-overexpressing MC38 (MC38-hCD47) colon adenocarcinoma cells.Nine days after tumor inoculation, we intravenously (i.v.) injected 64 Cu-hSIRPa-S36 K>R Nb into both groups.As additional control, the non-specific 64 Cu-GFP K>R Nb was injected in tumor-bearing hSIRPa/hCD47 mice.Non-invasive in vivo PET/MR imaging revealed a strongly enhanced 64 Cu-hSIRPa-S36 K>R Nb accumulation in the tumors of hSIRPa/hCD47 mice within the first minutes after injection, which remained stable at a high level for 6 h.In contrast, both control groups, 64 Cu-GFP K>R Nb-injected hSIRPa/ hCD47 mice and 64 Cu-hSIRPa-S36 K>R Nb-injected wt mice, showed rapid tracer clearance in the tumors and blood (Figure 5C).Importantly, 64 Cu-hSIRPa-S36 K>R Nb-injected hSIRPa/hCD47 mice exhibited a constantly higher PET signal in the blood over time, indicating a specific binding to circulating hSIRPa + myeloid cells (Figure 5C).Quantification of the PET images 3 h after injection revealed a significantly higher uptake in the tumors of hSIRPa/hCD47 mice (1.89 ± 0.09%ID/cc) compared with that of wt mice (0.60 ± 0.05% ID/cc) and to 64 Cu-GFP K>R Nb-injected hSIRPa/hCD47 mice (0.57 ± 0.05%ID/cc) (Figures 5C-E).Furthermore, we observed a ~7-fold enhanced uptake in the spleen, a ~2-fold enhanced uptake in the blood and liver, and a ~3-fold enhanced uptake in the salivary glands and bone in hSIRPa/hCD47 mice (Figures 5D, E), whereas no significant differences were identified in the kidney and the muscle tissue between the 64 Cu-hSIRPa-S36 K>R Nb-injected hSIRPa/hCD47 mice and both control groups (Figures 5D, E).From these results, we concluded that the novel 64 Cu-hSIRPa-S36 K>R Nb-based PET tracer is applicable to visualize and monitor the distribution of SIRPa + cells by non-invasive in vivo imaging.

Discussion
Myeloid cells, particularly macrophages, frequently infiltrate tumors, modulate tumor angiogenesis, promote metastasis, and have been associated with tumor resistance to chemotherapy and immune checkpoint blockade (27, 28).A characteristic marker for myeloid cells is the immune checkpoint SIRPa.Therapeutic targeting the SIRPa/CD47 signaling axis is considered a promising strategy for the treatment of advanced cancers.Recent in vivo data have demonstrated a synergistic anti-tumor effect of SIRPa-specific antibodies in combination with tumor-opsonizing antibodies such as cetuximab (EGFR), rituximab (CD20), and trastuzumab human epidermal growth factor receptor (HER2) (25, 26, 29), and, currently, several anti-hSIRPa monoclonal antibodies including BI 765063 and GS-0189 (FIS-189) are in clinical trials for mono-and combination therapies (30).In addition to serving as therapeutic target, SIRPa also represents a biomarker, which can be used to stratify patients by myeloid cell expression patterns (17-19) and to track the migration and dynamics of myeloid cells in the context of cancer.Recently, murine-specific SIRPa Nbs were successfully employed for noninvasive single-photon emission tomography imaging of myeloid cells in intracranial glioblastoma tumors of experimental mice (31).
Here, we pursued a binary screening strategy to develop the first hSIRPa-specific Nbs as a panel of novel theranostic binding molecules.Our aim was either to identify Nbs as modulating biologics blocking the hSIRPa/hCD47 axis or to monitor TAMs as the most common myeloid cell type in the TME.By choosing Nbs that exclusively bind the D1 domain of hSIRPa, we were able to identify binders that selectively block the interaction with CD47 and enhance ADCP in combination with the tumor-opsonizing antibody cetuximab in vitro.In particular, the selectivity of Nb S45 for binding hSIRPa, but not hSIRPg, might be advantageous, as recent data showed that nonselective hSIRPa/hSIRPg blockade can PET imaging with 64 Cu-hSIRPa-S36 K>R Nb. (A) Radiochemical purity of 64 Cu-hSIRPa-S36 K>R Nb was assessed using high-performance liquid chromatography (HPLC).(B) Antigen excess binding assay to determine the maximum binding (Bmax) of 64 Cu-hSIRPa-S36 K>R Nb, referred to as immunoreactive fraction. 64Cu-hSIRPa-S36 K>R Nb (1 ng) was applied to an increasing number of HT1080-hSIRPa cells of three technical replicates and binding curves were analyzed using the one-site nonlinear regression model.(C) Quantification of 64 Cu-hSIRPa-S36 K>R Nb tumor and blood uptake of s.c.MC38-hCD47 colon carcinoma-bearing hSIRPa/hCD47 mice over 6 h after injection. 64Cu-hSIRPa-S36 K>R Nb accumulation is compared to the control groups injected with control Nb or in MC38 wt mice injected with 64 Cu-hSIRPa-S36 K>R Nb.The resulting values were decay-corrected and presented as percentage of injected dose per cubic centimeter (%ID/cc).Representative data of one animal per group is shown.(D) Representative fused MIP (maximum intensity projection) PET/MR images of mice 3 h after 64 Cu-hSIRPa-S36 K>R (n = 4) or control Nb injection (each n = 4).PET signal in hSIRPa expressing myeloid cell-rich organs is compared to both control groups.Sites with increased 64 Cu-hSIRPa-S36 K>R Nb uptake are marked by colored arrows indicating the tumor (white and outlined), spleen (orange), bone (blue), salivary glands (purple), kidneys (green), and liver (red).In addition, axial sections of PET/MR images are shown where the tumors are highlighted with white circles and arrows.(E) Quantification of 64 Cu-hSIRPa-S36 K>R Nb in hSIRPa expressing myeloid cell-rich organs.High accumulation was also detected in sites of excretion, namely, the kidney and liver.The resulting values were decay-corrected and presented as percentage of injected dose per cubic centimeter (%ID/cc).Data are shown as individual plots and mean value (n = 4).p < 0.05 was considered statistically significant (*) and marked as ** for p < 0.01, *** for p < 0.001, and **** for p < 0.0001; non-significant results were marked with ns.
impair T-cell activation, proliferation, and endothelial transmigration (32).Notably, as versatile building blocks, Nbs can easily be customized into more effective biologics.Thus, blocking efficacies of the inhibitory hSIRPa-specific Nbs can be further improved, e.g., by establishing bivalent or biparatopic formats as previously shown (24, 33).Alternatively, bispecific binding molecules could be generated, e.g., by fusing the hSIRPa-blocking Nbs with a tumor-opsonizing Nb and Fc moiety (34,35) or CD40L expressed by activated T cells to bridge innate and adaptive immune responses (36).To address rapid renal clearance, which is a major drawback of small-sized Nbs for therapeutic application, other modifications such as PEGylation, addition of an albuminbinding moiety, or direct linkage to carrier proteins can be considered to extend their systemic half-life (t½) and efficacy (37,38).
In addition to developing inhibitory hSIRPa Nbs, we also identified binders to elucidate the presence and infiltration of the myeloid cell population using PET-based non-invasive in vivo imaging.Current diagnostic methods are based on histology and thus require biopsies through invasive sampling or endpoint analyses.These methods can be associated with severe side effects and limit the predictive value of such diagnostic approaches.In contrast, non-invasive in vivo whole-body molecular imaging techniques, particularly PET, represent a powerful method to monitor and quantify specific cell populations and thereby support individual therapy decisions (39)(40)(41).Because of their ideal characteristics for PET imaging, including specific binding, fast tissue penetration, and rapid renal clearance, Nbs emerged as next-generation tracer molecules with numerous candidates in preclinical and first candidates in clinical testing (42)(43)(44).With the hSIRPa-S36 Nb, we selected a functionally inert but highaffinity binding candidate for which we achieved site-directed chemical chelator labeling based on a unique protein engineering approach that did not compromise the stability or binding properties.Compared with other, more elaborate and less effective labeling strategies such as sortagging (45)(46)(47), this approach resulted in rapid chelator conjugation by applying straightforward NCS chemistry.64 Cu-hSIRPa-S36 K>R Nb-PET/MR imaging in a novel tumorbearing hSIRPa/hCD47 KI mouse model revealed rapid recruitment and sustained accumulation of our radiotracer in myeloid-enriched tumors and lymphatic organs with low background signal.We also observed a significantly enhanced 64 Cu-hSIRPa-S36 K>R Nb uptake in MC38-hCD47 adenocarcinomas of hSIRPa/hCD47 KI mice vs. wt mice, suggesting specific targeting of myeloid cells within the TME.This is also supported by the fact that no enhanced 64 Cu-hSIRPa-S36 K>R Nb uptake was observed in tumors and lymphatic organs of murine SIRPa and CD47 expressing wt mice.Beyond the crucial role of myeloid cells in tumor progression and cancer immunotherapy resistance, the occurrence of myeloid cells in diseased tissues is a hallmark of several inflammatory diseases like SARS-CoV-2 infection or autoimmune diseases such as systemic sclerosis, rheumatoid arthritis, and inflammatory bowel disease (48,49).Thus, the noninvasive in vivo monitoring of biodistribution, density, and dynamic changes of the myeloid cell compartment presented in this initial study would allow surveillance and early assessment of therapeutic response in a variety of diseases (50).In comparison to established strategies typically targeting TAM subpopulations visualizing the Translocator protein (TSPO) or the macrophage mannose receptor (MMR) using the 68 Ga anti-MMR Nb, the 64 Cu-hSIRPa-S36 K>R Nb enables the monitoring of the entire myeloid cell population (11,51,52).Furthermore, given that hSIRPa-S36 Nb detects both hSIRPa allelic variants, its application is not restricted to patient subpopulations.
In summary, this study demonstrates for the first time the generation and detailed characterization of hSIRPa-specific Nbs for potential therapeutic and diagnostic applications.Considering the important role of myeloid cells, particularly TAMs, the herein developed hSIRPa-blocking Nbs have the potential to extend current macrophage-specific therapeutic strategies (30,53).Moreover, our novel 64 Cu-hSIRPa-S36 K>R Nb-based PET tracer will broaden the growing pipeline of Nb-based radiotracers to selectively visualize tumor-associated immune cells by noninvasive in vivo PET imaging (45,47,51,54).Given the increasing importance of personalized medicine, we anticipate that the presented hSIRPa-specific Nbs might find widespread use as novel theranostics either integrated into or accompanying emerging immunotherapies.

Nanobody screening
For the selection of hSIRPa-specific Nbs, two consecutive phage enrichment rounds either with immobilized hSIRPa or hSIRPaD1 were performed.To generate Nb-presenting phages, TG1 cells comprising the Nb-library in pHEN4 were infected with the M13K07 helper phage.In each panning round, 1 × 10 11 phages were applied to streptavidin or neutravidin plates (Thermo Fisher Scientific) coated with biotinylated antigen (5 μg/mL).For biotinylation, purified antigen (Acrobiosystems) was reacted with Sulfo-NHS-LC-LC-Biotin (Thermo Fisher Scientific) in 5 M excess at ambient temperature for 30 min.Excess of biotin was removed by size exclusion chromatography using Zeba ™ Spin Desalting Columns 7K MWCO 0.5 mL (Thermo Fisher Scientific) according to the manufacturer's protocol.Blocking of antigen and phage was performed alternatively with 5% milk or Bovine Serum Albumin (BSA) in Phosphate-Buffered Saline with Tween (PBS-T), and, as the number of panning rounds increased, the wash stringency with PBS-T was intensified.Bound phages were eluted in 100 mM triethylamine (TEA) (pH 10.0), followed by immediate neutralization with 1 M Tris/HCl (pH 7.4).Exponentially growing TG1 cells were infected with eluted phages and spread on selection plates for subsequent selection rounds.In each round, antigenspecific enrichment was monitored by counting colonyforming units.

Whole-cell phage ELISA
For the monoclonal phage enzyme linked immunosorbent assay (ELISA) individual clones were picked, and phage production was induced as described above.Moreover, 96-well cell culture plates (Corning) were coated with poly-L-lysine (Sigma-Aldrich) and washed once with H 2 O. U2OS-wt and U2OS overexpressing hSIRPa (U2OS-hSIRPa) or hSIRPaD1 (U2OS-hSIRPaD1) were plated at 2 × 10 4 cells per well in 100 μL and grown overnight.The next day, 70 μL of phage supernatant was added to each cell type and incubated at 4°C for 3 h.Cells were washed five times with 5% FBS in PBS, followed by adding the Anti-M13 Monoclonal Antibody coulpled Horseradish Peroxidase (M13-HRP)-labeled detection antibody (Progen, 1:2,000 dilution) for 1 h, and washed three times with 5% Fetal Bovine Serum (FBS) in PBS.Finally, Onestep ultra TMB 32048 ELISA substrate (Thermo Fisher Scientific) was added to each well and incubated until color change was visible before stopping the reaction with 100 μL of 1 M H 2 SO 4 .For detection, the Pherastar plate reader at 450 nm was applied, and phage ELISA-positive clones were defined by a two-fold signal above wt control cells.

Biolayer interferometry
Analysis of binding kinetics of hSIRPa-specific Nbs was performed using the Octet RED96e system (Sartorius) as per the manufacturer's recommendations.In brief, biotinylated hSIRPa (5 μg/mL) diluted in Octet buffer (PBS, 0.1% BSA, and 0.02% Tween-20) was immobilized on streptavidin coated biosensor tips (SA, Sartorius) for 40 s.In the association step, a dilution series of Nbs ranging from 0.625 nM to 320 nM were reacted for 240 s followed by dissociation in Octet buffer for 720 s.Every run was normalized to a reference run applying Octet buffer for association.Data were analyzed using the Octet Data Analysis HT 12.0 software applying the 1:1 ligand-binding model and global fitting.For epitope binning, two consecutive association steps with different Nbs were performed.By analyzing the binding behavior of the second Nb, conclusions about shared epitopes were drawn.For the hCD47 competition assay, hCD47 was biotinylated and immobilized on SA biosensors followed by the application of pre-mixed solutions containing hSIRPa (20 nM) and Nb (250 nM).hCD47competing Ab KWAR23 (5 nM) was used as control.

Stability analysis
Stability analysis was performed by the Prometheus NT.48 (Nanotemper).In brief, freshly thawed hSIRPa Nbs were diluted to 0.25 mg/mL, and measurements were carried out at time point T 0 or after incubation for 10 days at 37°C (T 10 ) using highsensitivity capillaries.Thermal unfolding and aggregation of the Nbs were induced by the application of a thermal ramp of 20°C to 95°C while measuring fluorescence ratios (F350/F330) and light scattering.Via the PR.ThermControl v2.0.4, the melting temperature (T M ) and aggregation (T A gg ) temperature were determined.

PBMC isolation, cell freezing, and thawing
Fresh blood, buffy coats, or mononuclear blood cell concentrates were obtained from healthy volunteers at the Department of Immunology or from the ZKT Tübingen gGmbH.Participants gave informed written consent, and the studies were approved by the ethical review committee of the University of Tübingen, projects 156/2012B01 and 713/2018BO2.Blood products were diluted with PBS 1× (homemade from 10× stock solution, Lonza, Switzerland), and PBMCs were isolated by density gradient centrifugation with Biocoll separation solution (Biochrom, Germany).PBMCs were washed twice with PBS 1×, counted with a NC-250 cell counter (Chemometec, Denmark), and resuspended in heat-inactivated (h.i.) fetal bovine serum (Capricorn Scientific, Germany) containing 10% Dimethylsulfoxide (DMSO) (Merck).Cells were immediately transferred into a −80°C freezer in a freezing container (Mr. Frosty; Thermo Fisher Scientific).After at least 24 h, frozen cells were transferred into a liquid nitrogen tank and were kept frozen until use.For the experiments, cells were thawed in Iscove's Modified Dulbecco's Medium (IMDM) (+L-Glutamin + 25 mM (4-(2hydroxyethyl)-1-piperazineethanesulfonic acid) HEPES; Life Technologies) supplemented with 2.5% h.i.human serum (HS; PanBiotech, Germany), 1× Penicillin-Streptomycin (P/S) (Sigma-Aldrich), and 50 μm b-Mercaptoethanol (Merck), washed once, counted, and used for downstream assays.

In vitro radioimmunoassay
To determine the immunoreactive fraction (maximum binding, B max ), an increasing number of HT1080-hSIRPa cells were incubated in triplicates with 1 ng (2 MBq/μg) of 64 Cu-hSIRPa-S36 K>R Nb for 1 h at 37°C and washed twice with PBS/1% FBS.The remaining cell-bound radioactivity was measured using a Wizard² 2480 gamma counter (PerkinElmer Inc.) and quantified as percentage of the total added activity.

Analyses, statistics, and graphical illustrations
Graph preparation and statistical analysis were performed using the GraphPad Prism Software (version 9.0.0 or higher).One-way ANOVA was performed for multiple comparisons using Tukey as a post-hoc test (mean and SEM).A value of p < 0.05 was considered statistically significant and marked as * for p < 0.05, ** for p < 0.01, *** for p < 0.001, and **** for p < 0.0001; non-significant results were marked with ns.Graphical illustrations were created with BioRender.com.
The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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FIGURE 1 (B) Recombinant expression and purification of hSIRPa Nbs using immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC).Coomassie staining of purified Nbs is shown.(C) Stability analysis using nano-differential scanning fluorimetry (nanoDSF) displaying fluorescence ratio (350 nm/330 nm) and light intensity loss due to scattering shown as first derivative exemplarily shown for Nb S36 (upper panel).Data are shown as mean value of three technical replicates.BLI-based affinity measurements exemplarily shown for Nb S36 (bottom panel).Biotinylated hSIRPa was immobilized on streptavidin biosensors.Kinetic measurements were performed using four concentrations of purified Nbs ranging from 0.625 nM to 5 nM (displayed with gradually darker shades of color).The binding affinity (K D ) was calculated from global 1:1 fits shown as dashed lines.(D) Summary table of stability and affinity analysis of selected hSIRPa Nbs.Melting temperature (T M ) and aggregation temperature (T Agg ) determined by nanoDSF shown as mean ± SD of three technical replicates.Affinities (K D ), association constants (k on ), and dissociation constants (k off ) determined by BLI using four concentrations of purified Nbs shown as mean ± SD. (E) Representative images of hSIRPa and GFP-coexpressing U2OS cells stained with hSIRPa Nbs of three technical replicates.Images show individual Nb staining detected with anti-VHH-Cy5 (red), intracellular IRES-derived GFP signal (green), nuclei staining (Hoechst, blue), and merged signals; scale bar, 50 µm.

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FIGURE 2 Epitope characterization of hSIRPa Nbs.(A) Domain mapping analysis by immunofluorescence staining with hSIRPa Nbs on U2OS cells displaying human hSIRPa domain 1 (D1), domain 2 (D2), or domain 3 (D3) at their surface.Representative images of live cells stained with individual Nbs in combination with Cy5-labeled anti-VHH of three technical replicates are shown; scale bar, 50 µm.(B) Epitope binning analysis of hSIRPa Nbs by BLI.Graphical summary of epitope binning analysis on the different hSIRPa domains (left panel).Representative sensograms (n = 1) of combinatorial Nb binding to recombinant hSIRPa on sharing/overlapping epitopes or on different epitopes (right panel).

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FIGURE 3 Cross-reactivity and binding specificity of hSIRPa Nbs.(A) Cross-reactivity analysis of hSIRPa Nbs by immunofluorescence staining on U2OS cells displaying hSIRPa-V1, hSIRPa-V2, hSIRPb1, hSIRPg, or mouse SIRPa at their surface.Representative images of live cells stained with individual Nbs in combination with Cy5-labeled anti-VHH are shown of three technical replicates; scale bar, 50 µm.(B) Flow cytometry analysis of human peripheral blood mononuclear cells (PBMCs) stained with fluorescently labeled hSIRPa Nbs (AlexaFluor 647, AF647).Flow cytometry plots of Nb S36 staining on CD14 + and CD3 + PBMC populations derived from human donor K1 are shown as an example.(C) Flow cytometry analysis of hSIRPa Nbs staining CD14 + PBMCs of three different human donors (K1, K2, and K3).As control, PBMCs were stained with a Pep Nb (Control-Nb) and a SIRPa-antibody (positive control).Data are presented as mean ± SD of three technical replicates.

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FIGURE 4 Potential of hSIRPaD1 Nbs to augment phagocytosis of tumor cells.(A) Graphical illustration of hSIRPa/hCD47 interaction leading to suppression of macrophage-mediated phagocytosis of tumor cells.(B) Competition analysis of hSIRPa-binding to hCD47 in the presence of hSIRPaD1 Nbs (S12, S41, S44, and S45) by BLI (n = 1).Biotinylated hCD47 was immobilized on streptavidin biosensors, and a mixture of 20 nM hSIRPa and 250 nM of hSIRPaD1 Nbs or 5 nM of KWAR23 was applied to elucidate potential inhibition of hSIRPa binding to hCD47.(C) Schematic illustration of macrophage-mediated phagocytosis of tumor cells by hSIRPaD1 Nbs and tumor-opsonizing antibodies (e.g., the anti-EGFR antibody cetuximab).(D) Phagocytosis of CFSE-labeled DLD-1 cells by human monocyte-derived macrophages.A representative flow cytometry plot of the phagocytosis assay of cetuximab only and combinatorial treatment of cetuximab and hSIRPa Nb S45 with donor K1-derived macrophages is shown.(E) Quantitative analysis of the phagocytosis assay.Percent of phagocytosis of CFSE-labeled DLD-1 cells analyzed for macrophages derived from three different donors (K1, left; K2, center; K3, right) in different conditions is shown.Data are shown as individual and mean value of three technical replicates.p < 0.05 was considered statistically significant (*) and marked as ** for p < 0.01, *** for p < 0.001, and **** for p < 0.0001; nonsignificant results were marked with ns.